Physiology and Pathophysiology of Itch.

Itch is a topic to which everyone can relate. The physiological roles of itch are increasingly understood and appreciated. The pathophysiological consequences of itch impact quality of life as much as pain. These dynamics have led to increasingly deep dives into the mechanisms that underlie and contribute to the sensation of itch. When the prior review on the physiology of itching was published in this journal in 1941, itch was a black box of interest to a small number of neuroscientists and dermatologists. Itch is now appreciated as a complex and colorful Rubik's cube. Acute and chronic itch are being carefully scratched apart and reassembled by puzzle solvers across the biomedical spectrum. New mediators are being identified. Mechanisms blur boundaries of the circuitry that blend neuroscience and immunology. Measures involve psychophysics and behavioral psychology. The efforts associated with these approaches are positively impacting the care of itchy patients. There is now the potential to markedly alleviate chronic itch, a condition that does not end life, but often ruins it. We review the itch field and provide a current understanding of the pathophysiology of itch. Itch is a disease, not only a symptom of disease.

Eblasakimab, a novel IL-13 receptor alpha 1 monoclonal antibody, blocks STAT6 phosphorylation with low dose in human volunteers.

Eblasakimab is a first-in-class monoclonal antibody under investigation for the treatment of atopic dermatitis (AD), which targets IL-13Rα1, a subunit of the Type 2 receptor complex. IL-13Rα1 stimulates phosphorylation of signal transducer and activator of transcription 6 (STAT6) to drive inflammation. This brief report investigates the mechanistic basis of eblasakimab and its effects on IL-13Rα1 signaling as part of a phase 1a, open-label, single ascending dose study. Single ascending doses of eblasakimab were administered by intravenous or subcutaneous injection to healthy male volunteers. The impact of eblasakimab on IL-13Rα1 receptor occupancy and STAT6 phosphorylation was assessed in participant blood monocytes. No serious treatment emergent adverse events were reported. Eblasakimab effectively blocked the IL-13Rα1 receptor and inhibited STAT6 phosphorylation with single doses of 3 mg/kg intravenously and 300 mg subcutaneously. Results support further clinical development of eblasakimab as a novel biologic for AD, with potential for 2- to 4-week dosing regimens.

Mechanistic insights into the antipruritic effects of lebrikizumab, an anti-IL-13 mAb.

BACKGROUND: Atopic dermatitis is a chronic inflammatory skin disease with persistent and severe itch among its hallmark features. Significant increases in type 2 cytokines (ie, IL-4, IL-13, IL-31) have been documented in acute atopic dermatitis lesions and lead to multifaceted downstream effects, including inflammation, epidermal barrier dysfunction, and itch. OBJECTIVE: The primary objective of preclinical studies reported here was to test direct effects of IL-13 and an anti-IL-13 mAb, lebrikizumab, in a human dorsal root ganglion model in itch amplification, neuronal excitability, and transcriptional downstream targets. METHODS: Neuroactive effects were assessed via live cell calcium imaging, electric field stimulation, and RNA sequencing of human dorsal root ganglia stimulated with IL-13 alone or in combination with lebrikizumab. RESULTS: These preclinical findings suggest that IL-13 plays a direct enhancer role in multiple itch and neuroactive pathways as well as transcriptional downstream effects, and provide key insights into the mechanistic basis for lebrikizumab's anti-itch effects. CONCLUSION: IL-13 is a potent enhancer of neuronal responses to different itch stimuli, consistent with the neuroimmune axis contributing to chronic itch-associated inflammatory skin disease, and blockade of this cytokine pathway may provide a therapeutic approach.

Understanding the immune landscape in atopic dermatitis: The era of biologics and emerging therapeutic approaches.

Atopic dermatitis (AD) is a chronic, systemic, inflammatory disease that affects the skin and is characterized by persistent itch and marked redness. AD is associated with an increased risk of skin infections and a reduced quality of life. Most AD treatment options to date were not designed to selectively target disease-causing pathways that have been established for this indication. Topical therapies have limited efficacy in moderate-to-severe disease, and systemic agents such as corticosteroids and immunosuppressants present with tolerability issues. Advances in the understanding of AD pathobiology have made possible a new generation of more disease-specific AD therapies. AD is characterized by the inappropriate activation of type 2 T helper (Th2) cells and type 2 innate lymphoid (ILC2) cells, with a predominant increase in type 2 cytokines in the skin, including interleukin (IL)-13 and IL-4. Both cytokines are implicated in tissue inflammation and epidermal barrier dysfunction, and monoclonal antibodies targeting each of these interleukins or their receptors are in clinical development in AD. In March 2017, dupilumab, a human anti-IL-4Rα antibody, became the first biologic to receive approval in the United States for the treatment of moderate-to-severe AD. The anti-IL-13 monoclonal antibodies lebrikizumab and tralokinumab, which bind different IL-13 epitopes with potentially different effects, are currently in advanced-stage trials. Here, we briefly review the underlying pathobiology of AD, the scientific basis for current AD targets, and summarize current clinical studies of these agents, including new research to develop both predictive and response biomarkers to further advance AD therapy in the era of precision medicine.

Synergistic antipruritic effects of gamma aminobutyric acid A and B agonists in a mouse model of atopic dermatitis.

BACKGROUND: Despite recent insights into the pathophysiology of acute and chronic itch, chronic itch remains an often intractable condition. Among major contributors to chronic itch is dysfunction of spinal cord gamma aminobutyric acidergic (GABAergic) inhibitory controls. OBJECTIVES: We sought to test the hypothesis that selective GABA agonists as well as cell transplant-derived GABA are antipruritic against acute itch and in a transgenic mouse model of atopic dermatitis produced by overexpression of the TH2 cell-associated cytokine, IL-31 (IL-31Tg mice). METHODS: We injected wild-type and IL-31Tg mice with combinations of GABA-A (muscimol) or GABA-B (baclofen) receptor agonists 15 to 20 minutes prior to injection of various pruritogens (histamine, chloroquine, or endothelin-1) and recorded spontaneous scratching before and after drug administration. We also tested the antipruritic properties of intraspinal transplantation of precursors of GABAergic interneurons in the IL-31Tg mice. RESULTS: Systemic muscimol or baclofen are antipruritic against both histamine-dependent and -independent pruritogens, but the therapeutic window using either ligand alone was very small. In contrast, combined subthreshold doses of baclofen and muscimol produced a significant synergistic antipruritic effect, with no sedation. Finally, transplant-mediated long-term enhancement of GABAergic signaling not only reduced spontaneous scratching in the IL-31Tg mice but also dramatically resolved the associated skin lesions. CONCLUSIONS: Although additional research is clearly needed, existing approved GABA agonists should be considered in the management of chronic itch, notably atopic dermatitis.

Primary afferent and spinal cord expression of gastrin-releasing peptide: message, protein, and antibody concerns.

There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived "itch" neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord "itch" circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions.

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.

At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.

A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1.

BACKGROUND: Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE: We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS: We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS: Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION: IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice.

Chronic pancreatitis (CP) is a devastating disease characterized by persistent and uncontrolled abdominal pain. Our lack of understanding is partially due to the lack of experimental models that mimic the human disease and also to the lack of validated behavioral measures of visceral pain. The ligand-gated cation channel transient receptor potential ankyrin 1 (TRPA1) mediates inflammation and pain in early experimental pancreatitis. It is unknown if TRPA1 causes fibrosis and sustained pancreatic pain. We induced CP by injecting the chemical agent trinitrobenzene sulfonic acid (TNBS), which causes severe acute pancreatitis, into the pancreatic duct of C57BL/6 trpa1(+/+) and trpa1(-/-) mice. Chronic inflammatory changes and pain behaviors were assessed after 2-3 wk. TNBS injection caused marked pancreatic fibrosis with increased collagen-staining intensity, atrophy, fatty replacement, monocyte infiltration, and pancreatic stellate cell activation, and these changes were reflected by increased histological damage scores. TNBS-injected animals showed mechanical hypersensitivity during von Frey filament probing of the abdomen, decreased daily voluntary wheel-running activity, and increased immobility scores during open-field testing. Pancreatic TNBS also reduced the threshold to hindpaw withdrawal to von Frey filament probing, suggesting central sensitization. Inflammatory changes and pain indexes were significantly reduced in trpa1(-/-) mice. In conclusion, we have characterized in mice a model of CP that resembles the human condition, with marked histological changes and behavioral measures of pain. We have demonstrated, using novel and objective pain measurements, that TRPA1 mediates inflammation and visceral hypersensitivity in CP and could be a therapeutic target for the treatment of sustained inflammatory abdominal pain.

Serum kallikrein-8 correlates with skin activity, but not psoriatic arthritis, in patients with psoriatic disease.

BACKGROUND: About 30% of cutaneous psoriasis (PsC) patients develop psoriatic arthritis (PsA) in the joint, which is under-recognized by dermatologists. Biomarkers for PsA are needed so that early referral to a rheumatologist is made. Kallikreins (KLKs) are secreted serine proteases implicated in skin desquamation and inflammation. This study examined KLK potential as serum biomarkers of PsA in cutaneous psoriasis patients. METHODS: KLKs were measured by ELISAs in synovial fluids of three PsA patients and three control early osteoarthritis (OA) patients, as well as in a cohort of 152 serum samples collected from age- and sex-matched PsC patients, with (n=76) or without PsA (n=76). KLK expression in psoriatic plaques was examined by immunohistochemistry. Univariate and multivariate logistic regression analyses were conducted to analyze the association between serum KLK levels and disease class (PsC, PsA). Serum KLKs that associated with PsA were correlated with clinical parameters of skin and joint activity. RESULTS: Among the seven KLKs tested, KLK6 and KLK8 were elevated in both PsA synovial fluids and psoriatic plaques, but only serum KLK8 levels were associated with psoriatic disease (odds ratio=2.56, p=0.03). Although significantly elevated in PsC and PsA sera compared to healthy controls, KLK8 did not discriminate PsA from PsC patients. KLK8 correlated positively with the psoriasis area and severity index (PASI) (r=0.43, p=0.001) independent of age, sex and psoriasis duration ( ß=1.153, p=0.0003) and exhibited no correlations with tender or swollen joint counts. CONCLUSIONS: Increased KLK8 serum level in PsA patients reflects disease activity in the skin but not in the joints. Serum KLK levels are not useful for screening psoriasis patients for PsA.

Role of protease-activated receptors in human skin fibrosis and scleroderma.

Proteases and their receptors have poorly understood roles in skin fibrosis and systemic scleroderma (SSc). We examined the role of protease-activated receptors (PAR(1) and PAR(2) ) in the pathophysiology of human SSc and skin fibrosis. Immunohistochemistry showed that PAR(1) immunoreactivity was positive in fibroblasts of SSc skin and healthy skin. PAR(2) immunoreactivity was positive in SSc skin, but negative in endothelial cells and fibroblasts of healthy skin. Double immunofluorescence using an antibody against smooth muscle actin (α-SMA) as a marker for myofibroblasts verified a certain percentage of myofibroblasts positive for PAR(1) and PAR(2) in SSc skin. In human dermal cultured fibroblasts (HDF), PAR(1) stimulation with or without bleomycin pretreatment mobilized intracellular calcium, indicating that the expressed PARs are functional and have effects on downstream signalling by calcium release. PAR(2) -induced intracellular calcium mobilization was only measurable in HDF after bleomycin pretreatment. Thus, PAR(1) - and PAR(2) -positive fibroblasts are increased in SSc, indicating a regulatory role. Intriguingly, bleomycin activated PAR(2) in HDF indicating that fibrosis-promoting factors have a direct effect on PAR(2) expression and functionality.

Clinical, cellular, and molecular aspects in the pathophysiology of rosacea.

Rosacea is a chronic inflammatory skin disease of unknown etiology. Although described centuries ago, the pathophysiology of this disease is still poorly understood. Epidemiological studies indicate a genetic component, but a rosacea gene has not been identified yet. Four subtypes and several variants of rosacea have been described. It is still unclear whether these subtypes represent a "developmental march" of different stages or are merely part of a syndrome that develops independently but overlaps clinically. Clinical and histopathological characteristics of rosacea make it a fascinating "human disease model" for learning about the connection between the cutaneous vascular, nervous, and immune systems. Innate immune mechanisms and dysregulation of the neurovascular system are involved in rosacea initiation and perpetuation, although the complex network of primary induction and secondary reaction of neuroimmune communication is still unclear. Later, rosacea may result in fibrotic facial changes, suggesting a strong connection between chronic inflammatory processes and skin fibrosis development. This review highlights recent molecular (gene array) and cellular findings and aims to integrate the different body defense mechanisms into a modern concept of rosacea pathophysiology.

Neurovascular and neuroimmune aspects in the pathophysiology of rosacea.

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea.

Interferon-γ induces upregulation and activation of the interleukin-31 receptor in human dermal microvascular endothelial cells.

Interleukin-31 (IL-31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL-31 receptor complex for IL-31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon-γ (IFN-γ). Activation of the IL-31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN-γ-induced monokine induced by IFN-γ expression. Inhibitor studies revealed that the IFN-γ-induced IL-31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the T(H) 1-derived cytokine IFN-γ and the T(H) 2-derived cytokine IL-31 on endothelial cells.

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis.

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

Unilateral nephrectomy leads to up-regulation of syndecan-2- and TGF-beta-mediated glomerulosclerosis in syndecan-4 deficient male mice.

Syndecan-4 is an ubiquitous, plasma membrane-spanning heparan sulfate proteoglycan involved in proliferation, differentiation, adhesion and migration of cells in vitro. Syndecan-4 knockout (KO) mice show no obvious defects but respond abnormally to experimental stress conditions. In the adult, syndecan-4 is the most abundant syndecan of renal tissue. We therefore investigated the consequences of syndecan-4 deficiency during progression of kidney disease using unilaterally nephrectomized mice, a model of glomerular hyperfiltration and renal hypertrophy. 60 days after unilateral nephrectomy (UNX), mesangial expansion, enhanced matrix production (collagens I and IV, fibronectin) and focal segmental glomerulosclerosis, resembling early stages of diabetic nephropathy, was apparent in male but not female syndecan-4 KO mice. No defect was detected in wild type UNX males. Syndecan-2 mRNA and protein were not detectable in renal glomeruli of wild type mice, but were induced specifically in the glomeruli of the syndecan-4 deficient kidneys after unilateral nephrectomy. Due to the structural similarities of syndecans-2 and -4 we hypothesize that de novo-production of syndecan-2 in kidneys after unilateral nephrectomy reflects a compensatory response. However, this response is counterproductive since syndecan-2 supports the pro-sclerotic activity of TGF-beta1 which is increased in parallel with syndecan-2 synthesis. By contrast, signaling through syndecan-4 negatively controls the production of pro-sclerotic TGF-beta1.

Neuroimmune interactions in allergic skin diseases.

PURPOSE OF REVIEW: Recent studies have advanced our understanding that allergic inflammation triggers neuronal dysfunction, thereby modulating inflammation-related changes in affected tissues including the skin. Vice versa, evidence has emerged that inflammatory responses are controlled by neurons. Moreover, structural cells and invading immune cells express neuronal receptors and release mediators which directly communicate with nerve endings in the skin. RECENT FINDINGS: During the allergic response, skin cells do not only represent a significant source of neuromediators but also represent targets for neuropeptides or neurotrophins as well as neurotransmitters in the inflamed tissue. During the last decade, it has become obvious that a large variety of molecules influence the adaptive as well as the innate immune response. Beside neuropeptide receptors, proteinase-activated receptors, novel histamine receptors, different cytokine or chemokine receptors play a role in the pathophysiology of atopic and allergic diseases. SUMMARY: Peripheral sensory and autonomic nerves are critically involved in many pathways of the innate and adoptive immune system during allergic and atopic skin diseases. Further dissection of receptor-mediated and intracellular signal pathways will help to develop more effective therapeutic approaches for allergic and inflammatory skin diseases.

Involvement of TRPV4 in Serotonin-Evoked Scratching.

Several thermosensitive transient receptor potential channels (transient receptor potential vanilloid type-1, -3; transient receptor potential cation channel, subfamily A, member 1) have been implicated in itch. In contrast, the role of transient receptor potential vanilloid type-4 (TRPV4) in itch is unknown. Therefore, we investigated if TRPV4, a temperature-sensitive cation channel, plays an important role in acute itch in mice. Four different pruritogens, including serotonin (5-hydroxytryptamine [5-HT]), histamine, SLIGRL (protease-activated receptors 2/mas-related G-protein-coupled receptor C11 agonist), and chloroquine (mas-related G-protein-coupled receptor A3 agonist), were intradermally injected into mice and itch-related scratching behavior was assessed. TRPV4 knockout mice exhibited significantly fewer 5-HT-evoked scratching bouts compared with wild-type mice. Notably, no differences between TRPV4 knockout and wild-type mice were observed in the number of scratch bouts elicited by SLIGRL and histamine. Pretreatment with a TRPV4 antagonist significantly attenuated 5-HT-evoked scratching in vivo. Using calcium imaging in cultured primary murine dorsal root ganglion neurons, the response of neurons after 5-HT application, but not other pruritogens, was significantly lower in TRPV4 knockout compared with wild-type mice. A TRPV4 antagonist significantly suppressed 5-HT-evoked responses in dorsal root ganglion cells from wild-type mice. Approximately 90% of 5-HT-sensitive dorsal root ganglion neurons were immunoreactive for an antibody to TRPV4, as assessed by calcium imaging. These results indicate that 5-HT-induced itch is linked to TRPV4.